Citation for published version (APA): Sakamoto, K. (2002). Beer spoilage bacteria and hop resistance in Lactobacillus brevis Groningen: s.n.

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1 University of Groningen Beer spoilage bacteria and hop resistance in Lactobacillus brevis Sakamoto, Kanta IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2002 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Sakamoto, K. (2002). Beer spoilage bacteria and hop resistance in Lactobacillus brevis Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 The Membrane Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis Kanta Sakamoto, H. W. van Veen, Hiromi Saito, Hiroshi Kobayashi and Wil N. Konings This chapter was accepted to Applied and Environmental Microbiology. SUMMARY The activity of the membrane bound H + -ATPase of the beer-spoilage bacterium Lactobacillus brevis ABBC45 increases upon adaptation to bacteriostatic hop compounds. The ATPase activity is optimal around ph 5.6 and increases up to four fold when Lb. brevis was exposed to 666 µm of hop compounds. The extent of activation depends on the concentration of hop compounds and is maximal at the highest concentration tested. The ATPase activity is strongly inhibited by DCCD, a known inhibitor of F 0 F 1 -ATPase. Western blots of membrane proteins of Lb. brevis with the antisera raised against the α- and β-subunits of F 0 F 1 -ATPase from Enterococcus hirae show increased expression of the ATPase after hop adaptation. The expression levels as well as the ATPase-activity decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of Lb. brevis to hop compounds. 61

3 INTRODUCTION The hop plant, Humulus lupulus, L. is used in beer fermentation for its contribution to the bitter flavor of beer. Furthermore, the usage of hop in the brewing industry is preferred because hop has antibacterial activity and prevents beer from bacterial spoilage. Hop compounds are weak acids, which can cross cytoplasmic membranes in undissociated form in response to the transmembrane ph-gradient (Simpson and Smith, 1992). Due to the higher internal ph these compounds dissociate internally thereby dissipating the ph gradient across the membrane. As a result of this protonophoric action of hop compounds the viability of the exposed bacteria decreases (Simpson, 1993a, 1993b; Simpson and Smith, 1992). Some bacteria, however, are able to grow in beer in spite of the presence of hop compounds. Sami et al. (1997a) reported that Lactobacillus brevis ABBC45 strain could adapt to hop treatment and develop a high resistance to hop compounds. During hop resistance development the copy number of plasmid prh45 harboring hora gene increased (Sami et al., 1997a). Subsequent studies revealed that hora encodes a bacterial ATP-biding cassette (ABC) multidrug transporter (MDR) which can extrude hop compounds from the cell membranes upon ATP hydrolysis (Sakamoto et al., 2001). As a result of this exogenous expression of HorA in Lactococcus lactis, its resistance to hop compounds increased up to two fold. Micro-organisms have been found to increase the proton motive force (pmf)-generating activities in their cytoplasmic membranes when confronted with a high influx of protons (Viegas et al., 1998). The thermophilic bacterium Bacillus stearothermophilus (De Vrij et al., 1998) increases proton pumping respiratory chain activities when the proton permeability of its cytoplasmic membrane increases drastically at higher temperatures. In Enterococcus hirae (formerly Streptococcus faecalis) (Kobayashi et al., 1984, 1986) and Saccharomyces cerevisiae (Viegas et al., 1998) the proton translocating ATPase levels in their membranes were found to increase upon exposure to protonophores such as carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) or weak acids. Obviously, the main reason for this increase of the proton pumping activities is to maintain the pmf and the internal ph at viable levels. In view of the protonophoric activities of hop compounds it was of interest to investigate whether the hop-resistant Lb. brevis would respond in a similar way to the action of hop compounds and would increase the functional expression of its proton translocating ATPase in addition to the expression of the MDR HorA. In this study, we demonstrate that this is indeed the case and that Lb. brevis increases the functional expression of the proton translocationg ATPase during growth in the presence of hop compounds. 62

4 MATERIALS AND METHODS Bacterial strains and growth conditions Lactobacillus brevis ABBC45 was grown anaerobically at 30 C in MRS broth (Merck, Darmstadt, Germany). The initial ph of the growth medium was adjusted to 5.5 with HCl. Hop resistance and expression of HorA was achieved by growth of Lb. brevis in the presence of hop compounds, up to 666 µm, as described previously (Sami et al., 1997a). Cells grown in the presence of 666 µm of hop compounds were subcultured without hop compounds added in order to follow the ATPase activity under these growth conditions. Hop compounds A concentrated isomerized hop extract (Hopsteiner GmbH, Mainburg, Germany) was used as hop compounds. The iso-α-acid contents were determined, by using high-performance liquid chromatography (HPLC) (Rode et al., 1990). The concentration of hop compounds in the medium was expressed as the concentration of iso-α-acids. Preparation of the membrane Lb. brevis was grown to late exponential phase in the absence and in the presence of 100 µm and 666 µm hop compounds. Cells of Lb. brevis were harvested by centrifugation at 7,000 g for 15 min and washed twice at room temperature in 50 mm (K) HEPES (ph 7.4) containing 5 mm MgSO 4. The cells, suspended in the same buffer, were lysed at 37 C by treatment for 1.5 h with 1 mg/ml lysozyme (Sigma, USA) and 50 µg/ml mutanolysin (Sigma, USA) in the presence of a cocktail of proteinase inhibitors (Complete [Boehringer Mannheim, Germany] ). After the addition of DNase I (50 µg/ml) and RNase (1 µg/ml), the suspension was passed three times through an ice-cold French pressure cell at 70 MPa. Unbroken cells were subsequently removed by centrifugation at 7,000 g for 15 min at room temperature. The supernatant was centrifuged at 200,000 g for 45 min at 4 C and the pellet was suspended in the same buffer. This membrane fraction was used for ATPase assays and Western Blot analysis. The concentration of the membrane proteins was determined with D C protein assay kit (BioRad, USA) with bovine serum albumin as a quantitative standard. ATPase Assay ATPase activity was estimated from the release of inorganic phosphate measured by a modification of the method of Driessen et al. (1991). 1 or 2 µg of membrane protein was incubated at 30 C for 10 min in 50 mm (K) Mes buffer (usually at ph 5.5) containing 5 mm MgCl 2. ATP [Potassium salt] was added at a final 63

5 concentration of 2 mm to initiate the reaction. The reaction (total volume of 40 µl) was stopped after 5 min by immediately cooling the test tubes on ice. Malachite green solution (200 µl of 0.034%) was added, and after 40 min the color development was terminated by the addition of 30 µl citric acid solution (34% [w/v]). Immediately, the absorbance at 660 nm was measured with a multiscan photometer (Multiskan MS; Labsystems, Finland). One unit ATPase-activity was defined as the release of 1 µmole of inorganic phosphate in 1 min. Calibration was done by using a series of Pi standards (Sigma, USA). For the determination of ph dependency of the ATPase activity, membranes were incubated for 60 min on ice in 50 mm (K) Mes buffer, adjusted to various ph values. The ATPase activity was assayed at those different ph values as described above. To measure the effects of inhibitors on the ATPase activity, the membranes were pre-incubated with N, N - dicyclohexylcarbodiimide (DCCD; final concentration 0.2 mm), ortho-vanadate (final concentration 0.2 mm) or nitrate (K 2 NO 3 ; final concentration 25 mm) for 10 min at 30 C, and subsequently for 60 min on ice. The membrane sample without inhibitor was used as the control. Western Blotting Analysis The membrane protein of Lb. brevis, prepared as described above, was solubilised in Laemmli sample buffer containing 2% SDS (Laemmli, 1970) and separated by electrophoresis (20 µg of protein / lane) through 10% SDSpolyacrylamide gel by the method of Laemmli (1970). The protein bands were transferred to a polyvinilidene difluoride (PVDF) filter membrane and detected with the antisera raised against the F 1 complex of Enterococcus hirae H + -ATPase (Arikado et al., 1999) which can also bind with F 0 F 1 -ATPase from Lactococcus lactis (Amachi et al., 1998). Membranes from E. hirae prepared as previously described (Arikado et al., 1999) were used as control. The antibody-bound proteins were made visible with nitrobluetertazorium (NBT) and 5-bromo-4-chloro-3- indolyl phosphate (BCIP) (Gibco BRL. USA). The intensities of the bands were measured by densitometric analysis with NIH Image software v.1.61 (NIH, USA). 64

6 RESULTS Effect of hop on the ATPase activity In a previous publication it has been demonstrated that under these conditions Lb. brevis develops hop resistance by overexpressing the MDR HorA (Sami, 1999). The cytoplasmic membranes of cells were isolated as described in Materials & Methods and the ATPase activities in these membranes were determined as a function of the ph ranging from ph 4.4 to 7.0. All membranes of Lb. brevis grown in the presence of different levels of hop compounds showed maximum ATPase activity at around ph 5.6 (Fig. 1). At ph 5.6 membranes from the cells adapted to 666 µm of hop compounds had the highest activity, which was about 4-times the ATPase activity of membranes from non-adapted cells. The ATPase activities of membranes from cells adapted to 100 µm of hop compounds were in between these extremes and about 1.7 times the activity of the membranes from the non-adapted cells. Once the hop-adapted cells (666 µm) were subcultured in medium without hop compounds the ATPase activity of their membranes decreased rapidly (Fig. 1). ATPase activity (Pi µmol/min/mg protein) ph Figure 1. The ph profile of the ATPase activity in membranes of Lb. brevis. The ATPase activity at ph values ranging from 4.4 to 7.0 was measured of membranes prepared from cells grown without hop compounds (W0, ) and of cells adapted to 100 µm (W100, ) and to 666 µm of hop compounds (R666, ), and of cells deadapted by growth first in the presence of 666 µm of hop compounds followed by growth for two days in the absence of hop compounds (R0, ). The ATPase activity was shown as the amount of released inorganic phosphate (Pi) per min / mg protein. Effect of inhibitors on the ATPase activity To characterize the type of ATPase present in the membrane of Lb. brevis, the effect of several kinds of inhibitors on the ATPase activity was studied (Fig. 2). The ATPase activities of membranes from non-adapted cells and from cells adapted to different concentrations of hop compounds were all significantly inhibited by the F 0 F 1 -type inhibitor N, N -dicyclohexylcarbodiimide (DCCD). Moderate inhibition was observed with the P-type inhibitor ortho-vanadate, while the V-type inhibitor K 2 NO 3 showed the least inhibition or even activation in 65

7 membranes from cells grown at 100 µm of hop compounds (W100 in Fig. 2). These results correspond to the observations made for the enterococcal F 0 F 1 -type ATPase, which is slightly inhibited by ortho-vanadate and slightly enhanced by K 2 NO 3 (Y. Kakinuma, personal communication), indicating that F 0 F 1 -type ATPase is the major ATPase in membranes from Lb. brevis. ATPase activity (Pi µmol/min/mg protein) control DCCD vanadate nitrate W0 W100 R666 Figure 2. The effect of inhibitors on the ATPase activity of Lb. brevis. The ATPase activity of the membranes of W0, W100 and R666 (See the legend of Fig. 1) was measured at ph 5.6 in the presence of 0.2 mm of N, N - dicyclohexylcarbodiimide (DCCD) (closed bars), 0.2 mm of ortho-vanadate (vertically striped bars) or 25 mm of nitrate (horizontally striped bars). The activity without any inhibitor was also measured as control (open bar). Western Blot Analysis Two bands were strongly detected from the membranes of Lb. brevis with the antisera against the α- and β- subunits of the F 1 ATPase complex from E. hirae, which strongly indicates the F 0 F 1 -type nature of the ATPase of Lb. brevis. The apparent molecular weights of these bands are slightly higher than those of the α- and β- subunits of F 1 from E. hirae (Fig. 3A). The intensities of both bands are higher in membranes isolated from cells grown at higher concentrations of hop compounds and decrease again in membranes from hop-adapted cells (666 µm) subcultured in medium without hop compounds (Fig. 3B.). The intensities of both bands correlated well (r = 0.990; r = correlation coefficient) with the ATPase activities of the different membranes. The rate and extent of growth in MRS broth of hop adapted cells are slower than of non-adapted cells (Sami et al., 1997a). Also hop-adapted cells are smaller than cells grown in the absence of hop compounds (data not shown). 66

8 Figure 3. Western blot analysis of membranes of Lb. brevis and E. hirae with antisera against F 1 of E. hirae. Membranes of Lb. brevis were solubilized and separated by electrophoresis through 10% polyacrylamide gel (lanes 2-5). For comparison the results with membranes from E. hirae are shown in lane 1. The proteins were transferred to a PVDF filter membrane and reacted with the antisera raised against the F 1 complex of E. hirae H + -ATPase. (A) The result of Western Blotting: Lane1, E. hirae cultured at ph 6.0; Lane 2, Lb. brevis grown without hop (W0); Lane 3, Lb. brevis adapted to 100 µm of hop compounds (W100); Lane 4, Lb. brevis adapted to 666 µm of hop compounds (R666); Lane 5, Lb. brevis deadapted from 666 µm to 0 µm of hop compounds (R0). The arrows indicated the position of the α- or β-subunit of H + - ATPase from E. hirae. (B) The intensity of the lower bands of the ATPase from Lb. brevis. The intensities of these bands were measured with NIH Image software and presented in arbitrary units. α β Band Intensity (a.u.) A B W0 W100 R666 R0 (kda) DISCUSSION The beer spoilage bacterium Lb. brevis ABBC45 develops hop resistance upon growth in hop-containing media (Sami et al., 1997a). This resistance was found to be mediated by the functionally expressed multidrug resistance ABC transporter HorA (Sami, 1999; Sakamoto et al., 2001). Studies of HorA, functionally expressed in Lactococcus lactis, revealed that HorA can excrete the lipophilic hop compounds and several other MDR substrates from the membrane into the external medium (Sakamoto et al., 2001). Recently a second proton motive force-driven MDR with affinity for hop compounds has been found in Lb. brevis ABBC45 lacking HorA (Suzuki et al., 2002). The activity of HorA and this pmf-driven MDR thus results in a reduced influx of the undissociated and membrane permeable iso-α-acids into the cytoplasm and thereby limits the anti-bacterial pmfdissipating effect of hop compounds. Since Lb. brevis develops resistance against rather high concentrations of hop compounds, the question arose whether 67

9 functional expression of HorA and the pmf-driven MDR was sufficient to confer this resistance or whether additional activities could contribute to hop resistance. Anaerobic Gram-positive lactic acid bacteria such as Lb. brevis depend for the generation of their proton motive force strongly on their membrane bound H + -F 0 F 1 - ATPase (Kobayashi et al., 1986; Konings et al., 1995). In this study, we demonstrated that the functional expression of a membrane-bound H + -F 0 F 1 -ATPase increased during hop-resistance development and decreased again when the exposure to hop compounds was stopped. Previously, it was demonstrated that also the expression of the HorA transporter increased during hop-resistance development (Sami, 1999). The H + -F 0 F 1 -type nature of the ATPase was confirmed by H + -F 0 F 1 -ATPase effectors and especially by immunologial studies with the antisera against α- and β-subunits of H + -F 0 F 1 -ATPase from E. hirae. In accordance with the observations of Kobayashi et al. (Kobayashi et al., 1984, 1986) made in the anaerobic Gram-positive bacterium E. hirae, the increased functional expression of H + -F 0 F 1 -ATPase most likely allows Lb. brevis to maintain a viable pmf and intracellular ph in the presence of the protonophoric hop compounds. The results of this study together with those of previous reports (Sami, 1999; Sakamoto et al., 2001; Suzuki et al., 2002) indicate that Lb. brevis becomes resistant to hop compounds by the combined action of two ATP-driven systems: the H + -ATPase and the MDR pump HorA (Sami, 1999; Sakamoto et al., 2001), and a pmf-driven MDR (Suzuki et al., 2002). HorA and the pmf-driven MDR reduce the influx of the weak acidic hop compounds by pumping the undissociated hop compounds from the membrane environment into the external medium. The H + - ATPase compensates for the pmf-dissipating and internal ph-decreasing effects of hop compounds, which have escaped the MDR activities, by pumping more protons from the cytoplasm across the membrane. As a result of the higher expression of ATPase and of HorA and the energy dissipation by hop compounds the rate and extent of growth in MRS broth of hop adapted cells are slower than of non-adapted cells (Sami et al., 1997a). Those various hop resistance mechanisms (Fig. 4) are another demonstration of the versatility of bacteria and their capacity to recruit a variety of mechanisms to cope with toxic compounds in their environments. 68

10 Hop-H Hop-H Hop-H a Hop-H b ATP ADP H + ATP Hop - Hop - +H + c H + Mn 2+ ADP Hop-Mn-Hop Cytoplasmic membrane Cell wall H + Figure 4. Proposed mechanisms of hop resistance in Lb. brevis ABBC45 by the combined action of two ATP-driven systems and one proton motive force-driven MDR. The undissociated hop compounds (Hop-H) intercalate into the cytoplasmic membrane and are pumped out by the multidrug resistant ABC-type transporter HorA (a) (Sami, 1999; Sakamoto et al., 2001) and by a secondary MDR (b) (Suzuki et al., 2002). A fraction of Hop-H escapes the pumping activity of the transporters and enters the cytoplasm. In the cytoplasm Hop-H dissociates due to the higher internal ph into the anion (Hop ) and H +. H + also enters the cytoplasm in antiport with Hop-H by the secondary transporter. Hop may bind to cations such as Mn 2+ (Archibalt and Fridovich, 1981; Simpson, 1993a, 1993b; Simpson et al., 1993: Simpson and Hughes, 1993), while the increased H + -ATPase activity excretes H + across the membrane (c) (This work). 69

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